Apoptosis is a form of cell death that is carried out by a specialized intrinsic machinery. Although the knowledge about the biochemical mechanisms of apoptosis is fragmentary, it appears that signalling pathways that regulate apoptosis converge on a conserved machinery that disassembles a cell. An essential part of this machinery is a family of cysteine proteases related to interleukin-1xcex2-converting enzyme (ICE) (Lazebnik, Y. A., et al., Proc. Natl. Acad. Sci. USA, 92:9042-9046 (1995); (Lazebnik, Y. A., et al., Nature, 371:346-347 (1994); Nicholson, D. W., et al., Nature, 376:37-43 (1995); Yuan, J., et al., Cell, 641-652:641-652 (1993)). ICE-like proteases (ILPs) are expressed as inactive pro-enzymes that have to be activated to cleave their substrates and to contribute to apoptosis (Horvitz,. H. R., et al., Cold Spring Harb. Symp. Quant. Biol., 59:377-385 (1994); Oltvai, Z. N. and Korsmeyer, S. J., Cell, 79:189-192 (1994)).
Cancer chemotherapy kills cells by induction of apoptosis (Lowe, S. W., et al., Cell, 74:957-967 (1993); Fisher, D. E., Cell, 78:539-542 (1994)). Defects of apoptosis common in cancer cells contribute to tumor regression and appear to be responsible for the failure of current cancer chemotherapy. Alternatively excess apoptosis can lead to neurodegenerative diseases (e.g., Huntington""s Disease, Alzheimers Disease).
Therefore, more effective chemotherapeutic agents are needed.
The present invention relates to a method of identifying a compound (agent) which modulates (inhibits or activates) apoptosis in transformed cells. In one embodiment, the present invention is a method of identifying a compound that selectively activates apoptosis in transformed cells. In another embodiment, the present invention is a method of identifying a compound which inhibits apoptosis in cells.
The present method is carried out in a cell-free system. In the embodiment in which a compound that modulates apoptosis is identified, the following reagents are combined to produce a test sample: the compound to be assessed and a cellular extract that includes apoptotic machinery and a signal which activates, or is capable of activating, the apoptotic machinery. The combination is maintained under conditions appropriate for activation of apoptosis. Modulation of apoptosis is detected in the presence of the compound to be assessed by detecting or measuring modulation or a change in the activity of the apoptotic machinery of the cellular extract. Modulation of apoptosis indicates that the compound to be assessed modulates or affects apoptosis in transformed cells.
Modulation of apoptosis can be assessed by measuring interleukin-1xcex2-converting-enzyme like protease (ILP) activity. Compounds that inhibit apoptosis in transformed cells have a dose-dependent inhibitory effect on ILP activity, and compounds which activate apoptosis stimulate a dose dependent increase in protease activity.
In the embodiment in which a compound which selectively activates apoptosis in transformed cells is to be identified, the following reagents are combined to produce a test sample: the compound to be assessed; and apoptotic machinery and a signal which activates the apoptotic machinery (e.g., obtained from cellular extracts of transformed cells). The combination is maintained under conditions appropriate for activation of apoptosis; activation of apoptosis is detected in the presence of the compound to be assessed by detecting or assessing an event indicative of an increase in activity of the apoptotic machinery.
In the method in which a compound which selectively activates apoptosis in transformed cells is to be identified, an inhibitor of apoptosis can also be combined to produce the test sample. In this embodiment, the inhibitor inhibits or uncouples the apoptotic machinery of the cellular extract. Recoupling of the apoptotic machinery (partial or complete activation or reactivation of the ability of the signal to activate apoptosis) indicates that the compound to be assessed selectively activates apoptosis in transformed cells.
In the embodiment in which an inhibitor of apoptosis is to be identified, the following reagents are combined: a compound to be assessed and apoptotic machinery and a signal which activates, or is capable of activating, the apoptotic machinery. The combination is maintained under conditions appropriate for activation of apoptosis; inhibition of apoptosis is detected in the presence of the compound to be assessed by detecting uncoupling of the apoptotic machinery (partial or complete inhibition of the ability of the signal to activate the apoptotic machinery). Uncoupling of the apoptotic machinery indicates that the compound to be assessed inhibits apoptosis in cells.
The invention also relates to a method of selectively killing transformed cells, wherein the transformed cell is contacted with a compound which selectively activates apoptosis in transformed cells, as described herein. The invention also relates to methods of treating diseases associated with defective apoptotic machinery (e.g., cancer, neurodegenerative disease). In one embodiment, the invention relates to a method of treating cancer in an individual, wherein an effective amount of a compound which selectively activates apoptosis in transformed cells is administered to the individual. In another embodiment, the invention relates to a method of treating a neurodegenerative disease in an individual, wherein an effective amount of a compound which modulates apoptosis, as identified herein, is administered to an individual.
The methods of the present invention are useful for defining the biochemical mechanisms of apoptosis, and for the identification of agents that overcome the effects of known inhibitors of ILP activation. Given the central role that ILPs play in apoptosis, an understanding of the process of ILP activation provides a means by which apoptosis can be manipulated. In addition, the invention provides an assay to identify compounds which modulate (inhibit, activate) apoptosis, supplying new drugs which target defective apoptotic machinery associated with disease (e.g., cancer, neurodegenerative diseases such as Huntington""s Disease and Alzheimers Disease).
The invention further provides an alternative to current chemotherapy techniques. Current chemotherapy results in an increase of the apoptotic signal and inflicts cellular damage in cancer cells and normal cells. However, this approach fails when the signal required to exceed the threshold in drug-resistant cells is sufficient to overcome the threshold in untransformed cells. As described herein, transformed cells harbor a signal which is capable of activating the apoptotic machinery in transformed cells. This signal, which is absent in untransformed cells, is uncoupled from the apoptotic machinery in drug-resistant cells. Thus, in contrast to current chemotherapy, compounds identified as described herein, which recouple the signal to the apoptotic machinery, will selectively kill these cells, and result in a more effective treatment for cancer.